The halt in the extraction of wild marine resources has put a stop to fish flour availability worldwide and has increased prices (>1400 USD/ton) (International Monetary Fund). As a consequence the diets used in the production of aquiculture species like salmons have partially substituted this important consumable by other proteic ingredients among which the proteic consumables of vegetable origin stand out. Among these consumables the soybean meal, a sub-product of soy oil production, provides important advantages due to its low cost (<400 USD/Ton), its adequate proteic contents approximately 48% and a balanced amino acids profile (International Monetary Fund). Anyhow, its inclusion within the diets for salmons does not exceed 25% (replacement for fish meal) because higher rates of this consumable produce morphologic alterations of the salmon bowel which in turn produce a reduction in the productive parameters of the fish/salmonids (growth). These adverse effects have been attributed to the presence of different anti-nutritional factors (ANFs) where the thermostable ANFs stand out.
The soybean meal ANFs include some carbohydrates, like alpha-galactosides and non-starch polysaccharides (NSPs) (Choct, M.; rsjant-Li Y.; McLeish, J.; Peisker, M. 2010, Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritíve and Anti-nutritive Effects in Pigs and Poultry. Asian-Australasian Journal of Animal Sciences, 23, 1386-1398; Francis, G.; Makkar, H.; Becker, K. 2001, Anti nutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish, Aquaculture, 199, 197-227; Karr-Lilienthal, L. K.; Kadzere, C. T.; Grieshop, C. M.; Fahey, G. C. 2005, Chemical and nutritional properties of soybean carbohydrates as related to no ruminants: A review. Livestock Production Science, 97, 1-12). The main alpha-galactosides contained in the soybean meal are the stachyose and the raffinose and its concentrates ranges from 2 and 5% w/w and 0.5 and 2% w/w of dry product respectively. (Choct, M.; rsjant-Li Y.; McLeish, J.; Peisker, M. 2010, Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritive and Anti-nutritive Effects in Pigs and Poultry. Asian-Australasian Journal of Animal Sciences, 23, 1386-1398; Karr-Lilienthal, L. K.; Kadzere, C. T.; Grieshop, C. M.; Fahey, G. C. 2005, Chemical and nutritional properties of soybean carbohydrates as related to no ruminants: A review. Livestock Production Science, 97, 1-12). The total concentration of NSPs in the soybean meal is of around 15-20% of dry weight. This term (NSPs) groups three different types of polysaccharides: cellulose, hemicelluloses and pectins (Huisman, M. M. H.; Schols, H. A.; Voragen, A. G. J. 1998, Cell wall polysaccharides from soybean (Glycine max.) meal. Isolation and characterization. Carbohydrate Polymers, 37, 87-95; Karr-Lilienlhal, L. K.; Kadzere, C. T.; Grieshop, C. M.; Fahey, G. C. 2005, Chemical and nutritional properties of soybean carbohydrates as related to no ruminants: A review. Livestock Production Science, 97, 1-12; Knudsen, K. E. B. 1997, Carbohydrate and lignin contents of plant materials used in animal feeding. Animal Feed Science and Technology, 67, 319-338).
The deleterious of the ANFs was documented both for the alpha-galactosides and the NSPs in different animal species. In pigs, the NSPs and alpha-galactosides supplement has shown an adverse effect in growth (Choct, M.; rsjant-Li Y.; McLeish, J.; Peisker, M. 2010, Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritive and Anti-nutritive Effects in Pigs and Poultry, Asian-Australasian Journal of Animal Sciences, 23, 1386-1398; Karr-Lilienthal, L. K.: Kadzere, C. T.; Grieshop, C. M.; Fahey, G. C. 2005, Chemical and nutritional properties of soybean carbohydrates as related to no ruminants: A review. Livestock Production Science, 97, 1-12). In poultry, the NSPs alter the digestion of nutrients because the lack of digestive enzymes and the soluble part of the NSPs create a viscous condition in the small intestine, altering the activity of the digestive enzymes. The alpha-galactosides reduce the body weight, the average daily weight gain and the feed conversion ratio (Choct, M.; rsjant-Li Y.; McLeish, J.; Peisker, M. 2010, Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritive and Anti-nutritive Effects in Pigs and Poultry. Asian-Australasian Journal of Animal Sciences, 23, 1386-1398; Karr-Lilienthal, L. K.; Kadzere, C. T.; Grieshop, C. M.; Fahey, G. C. 2005, Chemical and nutritional properties of soybean carbohydrates as related to no ruminants: A review. Livestock Production Science, 97, 1-12). In aquaculture species the NSPs and alpha-galactosides are also considered anti nutritional factors (Francis, G.; Makkar, H.; Becker, K. 2001, Anti nutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, 199, 197-227). Their presence in the diet of salmons increase the contents of water and minerals in the feces and block up the action of digestive enzymes (Francis, G.; Makkar, H.; Becker, K. 2001, Anti nutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, 199, 197-227).
In aquaculture the situation is critical because the aquaculture species such as salmon have higher protein requirements than poultry or pig (Wilson, R.; John, H.; Hardy, R 2002. Amino Acid and Proteins. in: H. John, R. Hardy (Eds.), Fish Nutrition. Academic Press, 3rd Edition, pp. 143.179) and at present the replacement of fish meal using other protein ingredients is a challenge for this industry (Gatlin, D.; Barrows, F.; Brown, P.; Dabrowski, K.; Gaylord, T.; Hardy, R; Herman, E.; Hu, G.; Krogdahl., A.; Nelson, R; Overturf, K.; Rust, M; Sealey, W.; Skonberg, D.; Souza, J.; Stone, D.; Wilson, R.; Wurtele, E. 2007, Expanding the utilization of sustainable plant products in aqua feeds: a review. Aquaculture Research, 38, 551-579; Tacon, A. G. J.; Metian, M. 2008, Global overview on the use of fish meal and fish oil in industrially compounded aqua feeds: Trends and future prospects. Aquaculture, 285, 146-158).
There are reduction alternatives by aqueous or alcoholic extraction of the ANFs which generate a product known as soy protein concentrate which increases its protein contents in approximately 65% as compared to the soybean meal. By including this product in fish diets the productive levels increase because it increases the digestion of nitrogen or amino acids, it increases the growth ratio and the feed conversion ratio. (Choct, M.; rsjant-Li, Y.; McLeish, J.; Peisker, M. 2010. Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritive and Anti-nutritive Effects in Pigs and Poultry. Asian-Australasian Journal of Animal Sciences, 23, 1386.1398; Olli, J. J., Krogdahl, A.; Vandeningh, T. S. G. A.; Brattas, L. E. 1994, Nutritive-Value of 4 Soybean Products in Diets for Atlantic Salmon (Salmo-Salar, L). Acta Agriculturae Scandinavica Section A-Animal Science, 44, 50-60). Nevertheless the cost of these consumables is similar to the one of fish meal restricting its use by the salmon culture industry. (Gatlin, D.; Barrows, E; Brown, P.; Dabrowski, K.; Gaylord, T.; Hardy, R; Herman, E.; Hu, G.; Krogdahl, A.; Nelson, R.; Overturf, K.; Rust, M.; Sealey, W.; Skonberg, D.; Souza, J.; Stone, D.; Wilson, R.; Wurtele, E. 2007, Expanding the utilization of sustainable plant products in aqua feeds: a review. Aquaculture Research, 38, 551-579). Another technology is the incorporation of glucohydrolases in the animal feed, although the effects of this alternative in productive trials have not produced concluding results in poultry, pigs or salmons (Bhat, M. K. 2000, Cellulases and related enzymes in biotechnology. Biotechnol. Adv., 18, 355-383; Caeter, C. G.; Houlihan, D. F.; Buchanan, B.; Michell, A. 1. 1994, Growth and feed utilization efficiencies of seawater Atlantic salmon, Salmo salar L, fed a diet containing supplementary enzymes. Aquaculture Research, 25, 37-46; Choct, M.; rsjant-Li, Y.; McLeish, J.; Peisker, M. 2010. Soy Oligosaccharides and Soluble Non-starch Polysaccharides: A Review of Digestion, Nutritive and Anti-nutritive Effects in Pigs and Poultry. Asian-Australasian Journal of Animal Sciences, 23, 1386-1398). The inclusion of cellulases, hemicellulases and pectinases in salmon feeds poses an important restriction regarding temperature. This is because the body temperature of the salmon corresponds to the aquatic environment in which they are, in Chile is 10-12° C. In contrast, the activity optimal temperature of these enzymes is 50° C. (Bhat, M. K, 2000, Cellulases and related enzymes in biotechnology. Biotechnol. Adv., 18, 355-383; Caeter, C. G.; Houlihan, D. F.; Buchanan, B.; Michell, A. I. 1994, Growth and feed utilization efficiencies of seawater Atlantic salmon, Salmo salar L., fed a diet containing supplementary enzymes. Aquaculture Research, 25, 37-46).
Biotechnological methods such as fermentation with bacteria, yeasts and fungi in solid state for the lignocellulosic biodegradation of agricultural sub-products such as soybean meal have also been proposed (Graminha, E. B. N.; Gongalves, A. Z. L.; Pirota, R. D. P. B.; Balsalobre, M. A. A.; Da Silva, R.; Gomes, E. 2008, Enzyme production by solid-state fermentation: Application to animal nutrition. Animal Feed Science and Technology, 144, 1-22).
The fermentation of soybean meal is a process that allows the decrease or the degradation of different anti-nutritional factors producing a consumable which use in animal production (like salmons, poultry and pigs) could offer important benefits over the soybean meal without fermenting. This situation would be especially attractive considering the possibility of degrading the thermostable ANFs. In this regards different microorganisms and types of fermentations have been proposed for the reduction of anti nutritional factors. For example fermentations with Debaryomyces hansenii or Lactobacillus brevis in which the degradation of alpha-galactosides (GOSs) has been assessed (Refstie, S. Sahlstrom; S., Brathen, E.; Baeverfjord, G.; Krogedal, P. 2005, Lactic acid fermentation eliminates indigestible carbohydrates and anti nutritional factors in soybean meal for Atlantic salmon (Salmo salar). Aquaculture, 246, 331-345; Rodrigues Brasil, A; Tabarez de Rezende, S.; do Carmo Gouveia, M.; Guimaraes, V. 2010, Removal of oligosaccharides in soybean flour and nutritional effects in rats, Food Chemistry, 118, 251-255). On the other hand fermentations with Aspergillus oryzae fungi or with Bacillus subtillis have been developed for the reduction of lectins or tripsin inhibitor (Hong, K.-J.; Lee, C.-H.; Kim, S. W. 2004, Aspergillus oryzae GB-107 Fermentation Improves Nutritional Quality of Food Soybeans and Feed Soybean Meals. Journal of Medicinal Food, 7(4), 430-435; Kim, S. W.; van Heugten, E.; Ji, F.; Lee, C. H.; Mateo, R. D. 2010, Fermented soybean meal as a vegetable protein source for nursery pigs: I. Effects on growth performance of nursery pigs. Journal of Animal Science, 88, 214-224; Wang, J. P.; Liu, N.; Song, M. Y.; Qin, C. L.; Ma, C. S. 2011, Effect of enzymolytic soybean meal on growth performance, nutrient digestibility and immune function of growing broilers. Animal Feed Science and Technology, 169 (3-4), 224-229).
There is a need for developing a process that may allow the obtention of a product suitable for feed, particularly for animals, more particularly for fish, especially for salmons that contains high contents of protein higher than 50% a balanced amino-acid profile and low contents of ANFs.
Soy bean fermentation for human feed purposes is a millenary procedure in Asian cultures like China, Japan, Thailand and India, among others. The fermentation can be made directly on ground soy or to its by-products like the soybean meal or soy milk and tofu, a curdle of soy milk. The fermentation of soy curdle (tofu) is produced by a fermentation in solid state with some strains of fungi like Actinomuco sp, Mucorwutungkino sp, Mhimelis sp, and Thizopuz sp. There are commercial strains that correspond to Actinomuco. The process is performed at a temperature between 25° C. and 30° C., anyhow in warm summers the strains of Thizopus oligosporus are more suitable because they grow at temperatures like 40° C. In China a soy fermentum with Aspergillus and Mucor strains or with bacterial strains is also produced and is known as douchi or touchi. In Japan this product is known as natto and there are three different products: Otohiki-natto which is made based on an inocculate of Bacillus natto which is a variant of Bacillus subtilis. In these cases the fermentation is done at a temperature between 40-45° C. On the other hand there is the yuki-wari which corresponds to a mixture of the former with rice, inoculated with koji. Koji means a “fungi lint” and it generally corresponds to the growth on an Aspergillus oryzae and Aspergillus sojae grain in a 20 days fermentation at 25° C.-35° C. Finally, the third product is known as the hama-natto which corresponds to a mixture of soy with rice, wheat and barley inoculated with koji, then an aging of one year in pressure is done.
The fermentum of soy pastes in Asia is known as miso. The process also contains a mixture of soy bean soaked in water and then boiled. This soy paste is mixed with koji and with other strains of fungi and bacteria like: Zygosaccharamyces rouxii, Torulopsis, Pediococcus, Halophilus and Streptococcus faecalis. This paste is known as green miso and corresponds to an anaerobic fermentation at temperatures of 25° C.-30° C., and it undergoes a period of aging that varies from one week in the case of “white miso” up to months or a year. This product is frequently used in the preparation of soups. Another very important Asian fermented product from soy is the soy sauce. There are at least 5 types of soy sauces recognized in Japan and two production processes are described that vary in the type of fermentation, one with an aerobic fermentation and another one with anaerobic fermentation. The most classic process is the aerobic which starts from the soybean meal without oil. This is soaked and dried, cooked at 130° C. during 45 minutes and mixed with roasted barley meal. This mixture is then inoculated with koji, brine is added and left to ferment for some days. Later there is an aging in controlled conditions of salt and temperature being the latter of 35° C.-40° C. during a 2 to 4 months period. This widely known processes are not related to the process or the product of this invention which uses a specific combination of four cellulolytic bacteria to perform a solid state fermentation of the soybean meal to decrease the anti-nutritional factors.
The document WO 2009065722 presents a method to ferment a substrate that contains a soy protein. The method comprises the steps of providing a sterile aqueous fluid containing 0.5 to 8% per weight of dissolved soy protein, 0 to 0.2% per weight of dairy protein and less than 24% per weight of solids; inoculating a fluid with a culture comprising bacteria from the group of selected lactic or acid lactic bacteria consisting of mesophilic Lactococcus, Leuconostoc, Lactobacillus (with optimal temperature under 35° C.) and combinations thereof; fermenting the inocculatid fluid by incubating at 20° C.-40° C. during 0.5 to 11 hours; wherein during the fermentation the following changes in the concentrations occur: the diacetil concentration increases in at least 0.2 ppm and/or the acetaldehyde concentration increases by at least 0.1 ppm; the concentration of at least an n-alcanal C5-C9 decreases at least by 30% and/or the concentration of trans-2-hexenal decreases in at least by 30%. The document is not related to the fermentation of soybean meal to the decrease of anti-nutritional factors using cellulolytic bacteria.
The document WO 2005032568 describes a soy product fermented by lactic acid fermentation that has a strong immunopotentiator effect and a favourable flavour and a process to produce it. The product is produced fermenting soy and a processed soy product through a co-culture of lactic acid bacteria with a yeast. The lactic acid bacteria is at least Enterococcus faecalis, optionally combined with another coccus, bacilli or bifidobacteria. The yeast is Saccharomyces cerevisiae and/or Saccharomyces rosei. The fermented soy is produced fermenting soy milk using the mentioned microorganisms, to provide a liquid fermented product that is then neutralized with a calcium compound and later dried to result in a powder fermented product. The document is not related to the fermentation of soybean meal to decrease anti-nutritional factors using cellulolytic bacteria.
The document WO 2002085131 presents a method to produce a tasty product from a protein source using a combination of two different strains of bacteria. The protein source can be soy, wheat or rice but it is preferable milk or whey. The first strain is selected among Macrococcus, Micrococcus, Enterococcus, Staphylococcus, Brevibacterium, Anthrobacter and Corynebacterium, preferably Macrococcus caseolyticus. The second strain is selected among Lactococcus, Lactobacillus, Pediococcus or Leuconostoc. The source of protein is fermented with the bacteria at a pH above the isoelectric point of the protein, preferably at a pH of 5.5 to 6.5. The document is not related to the fermentation of soybean meal to decrease anti-nutritional factors using cellulolytic bacteria.
The document CN102210412 describes a compound feed to improve the meet ratio of the Tilapia mossambica and a method to prepare it. The compound feed mainly contains fermented rapseed meal and fermented soybean meal and is effectively absorbed during the digestion. Both fermented meals are available in the market therefore detail of the fermentation process used are not included. The document informs that the fermentation process eliminates tannins, trypsin inhibitors, and other anti-nutritional factors. Said document does not disclose a way to do the fermentation since the fermented meals used are available in the market.
The above mentioned documents are the closest documents to the present invention, anyhow the previous state of the art does not consider favouring the nutritional value of soybean meal an issue to be solved by decreasing the anti-nutritional agents like non-starch polysaccharides and alpha-galactosides. For that reason the state of the art does not consider that, in order to obtain this improvement, a combination of the three types of bacteria this invention proposes can be used and to be used in a soybean meal solid state fermentation procedure.
In addition to the documents of the mentioned patents, in the state of the art some industrial fermentation processes for soybean meal are known. Hamlet Protein AS, Horsens, Denmark sells fermented soybean meal through a biotechnological process with a product presenting a reduction in the contents of oligosaccharides an increase in the concentration of proteins and the elimination of anti-nutritional factors. The process this company uses is not completely clear, although in their patent applications US20060233913, WO 2011147923 and US20110034394A1 processes for soy, other seeds and yeast proteins fermentation are described by means of yeasts or specific enzymes. The use of cellulolytic bacteria to prepare the products of this company is not mentioned.
Dongguan Yinhua Biotechnology Co. Ltd, Dongguan, China also sells fermented soybean meal. The process they use to treat the soybean meal is not also completely clear, although in their patent application CN101161810 they disclose they do fermentation of soy and other seeds using yeasts. They do not mention the use of cellulolytic bacteria to prepare the products of this company.
Unlike that disclosed in the state of the art, the process of the invention does not use industrial or market available enzymes. It uses selected bacteria that supply their enzymes during the growth on the soybean meal.